The chemical synthesis of nucleic acids involves step-wise assembly of suitable, protected phosphoramidite building blocks on solid phase, followed by the removal of protecting groups and the detachment from the solid phase. In addition to the incorporation of ribo- or deoxyribonucleotides, this method allows the introduction of further compounds including nucleotide analogs (containing modified sugar moieties and/or modified nucleobase moieties) as well as other compounds which allow specific detection (e.g. dyes) or specific binding (e.g. biotin). Ideally, the methods for introduction and final deprotection of these further compounds would be identical to methods used for the preparation of the unmodified (physiological) ribo and deoxribo oligonucleotides (RNA and DNA).
However, a variety of useful and routinely employed dyes and reporter groups are not stable under the conditions of assembly and deprotection of oligonucleotides and are therefore introduced post-synthetically by so-called conjugation reactions. Conjugation reactions are also carried out for the immobilization of nucleic acids on surfaces or beads. Such reactions require the presence of a specific chemical group on the oligonucleotide, which has a unique reactivity towards suitably activated dyes/binders or surfaces/beads. These specific chemical groups are usually amino or thiol groups and the materials, required for their introduction into oligonucleotides are called “amino linkers” or “thio linkers”, respectively. They typically contain a phosphorous activating group such as a phosphoramidite moiety for the attachment to the nucleic acid, a linker (e.g. alkyl chains or oligoethylenglycol chains of various lengths) and the suitably protected amino or thiol group, respectively.
The presently known amino linker compounds can be divided into two groups. In the first group, the amino protecting group comprises an acid labile mono- or dimethoxytrityl group. These acid labile groups can either be removed before or after removal of the other protecting groups which is carried out by ammonia or various amines. In the latter case, purification of the amino-linker modified nucleic acid can be achieved based on the lipophilic properties of the still present trityl group. Therefore, if purification based on lipophilic properties is desired, amino linkers of this first group are chosen.
The second group of amino linkers is characterized by protecting groups which are cleaved during the deprotection step together with the other protecting groups with ammonia or various amines. Advantageously, for linkers of the second group, no additional deprotection steps are required, and therefore the related manufacturing process is more straightforward. Consequently, they are employed on a much greater scale. In particular, these amino linkers of the second group are used in the high-volume routine and parallel synthesis of amino-modified nucleic acids. The most commonly employed and commercially available amino linkers in this group contain an amino group protected by a trifluoroacetyl group (Formula I), but other protecting groups, such as phthalimide (e.g. Formula XI, see FIG. 3) have been reported [U.S. Pat. No. 7,164,014, Huang et al.].
All known amino linkers of this second type are honey-like, viscous oils. As a consequence they are difficult to handle. For example they can only be purified by chromatography, they are difficult to aliquot in accurate portions and they are difficult to dry. Furthermore, they are quite unstable substances, because they are not stabilized by crystal packing and easily degrade by reaction with oxygen or water. As a consequence, they have to be shipped and stored at low temperatures.

Thus, it is an object of the present invention to provide protected linker compounds which overcome disadvantages of this second group of amino linker compounds available in the prior art and which are fully compatible with the standard assembly and deprotection protocols for nucleic acid synthesis. More particular objects of the present invention include to provide such amino linker building blocks which can be purified by precipitation or crystallization, which are easily aliquoted and which do not easily degrade by reaction with oxygen and water.
In a first aspect of the invention, protected amino linker compounds and more particularly protected amino linker phoshoramidite building blocks are provided. In a second aspect, methods of production of amino linker compounds are provided. In a third aspect of the invention, methods of adding a protected amino linker phoshoramidite building block to a previously assembled, protected and immobilized ribo- or deoxyribonucleic acid molecule or to a derivative thereof and methods of deprotecting the protected amino group of the added amino linker compound are provided. The third aspect of the invention also relates to amino functionalized nucleic acids or nucleic acid derivatives comprising an amino protecting group. In a fourth aspect, a use of a diamide substituted protecting group for protecting an amino group is provided. It is in particular the use of a 1,4-diamide group which is covalently bound to an aromatic system, in which two neighbouring C-atoms of an aromatic system are a 2- and a 3-position of the 1,4-diamide amino protecting group (for a detailed definition see Formula II in FIG. 1).
Additional objects and advantages of the invention are set forth in, or will be apparent to those of ordinary skill in the art, from the detailed description as follows. Also, it should be further appreciated that modifications and variations to the specifically illustrated and discussed features and materials hereof may be practiced in various embodiments and uses of this invention without departing from the spirit and scope thereof, by virtue of present reference thereto. Such variations may include, but are not limited to, substitutions of the equivalent means, features, and materials for those shown or discussed, and the functional or positional reversal of various parts, features, method steps, or the like.
Still further, it is to be understood that different embodiments, as well as different presently preferred embodiments, of this invention may include various combinations or configurations of presently disclosed features, elements, method steps, or their equivalents (including combinations of features or configurations thereof not expressly shown in the figures or stated in the detailed description).
These and other features, aspects, and advantages of the present invention will become better understood with reference to the following descriptions and the appended claims. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention, and, together with the descriptions, serve to explain the principles of the invention.